Protein Structure: Xray Crystallography

1
Protein Structure X-ray Crystallography

• Microbiology 343
• David Wishart
• david.wishart_at_ualberta.ca

2
Objectives

• To review the basics of protein structure
  (primary, secondary, supersecondary, tertiary)
• Understand the basic principles and steps in
  protein crystallization and protein
  cyrstallography
• Become aware of databases (PDB) and tools to help
  visualize proteins

3
Much Ado About Structure
Structure Function Structure
Mechanism Structure
Origins/Evolution Structure-based Drug
Design Solving the Protein Folding Problem
4
Protein Structures Are Complex
5
Protein Structure (A Review)
6
Ramachandran Plot
7
Secondary Structure
8
Beta Sheet
9
Alpha Helix
10
Reverse Turn
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Supersecondary Structure
12
Supersecondary Structure
13
Tertiary Structure
14
Proteins are Complex

• Average residue contains 8 heavy atoms
• Average protein contains 300 amino acids
• Average structure contains 2400 atoms
• First structure (sperm whale myoglobin) took 5
  years with a team of 15 key punch operators
  working around the clock to solve
• Most structures still take 1 year to solve

15
Solving Protein Structures

• Only 2 kinds of techniques allow one to get
  atomic resolution pictures of macromolecules
• X-ray Crystallography (first applied in 1961 -
  Kendrew Perutz)
• NMR Spectroscopy (first applied in 1983 - Ernst
  Wuthrich)

16
X-ray Crystallography
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X-ray Crystallography

• Crystallization
• Diffraction Apparatus
• Diffraction Principles
• Conversion of Diffraction Data to Electron
  Density
• Resolution
• Chain Tracing

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Crystallization
Protein Crystal
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Crystallization
20
Crystallization

• Start with a solution of the protein with a
  fairly high concentration (2-50 mg/ml)
• Add reagents (PEG) that reduce the solubility
  close to spontaneous precipitation
• Perform further concentration slowly until small
  crystals may start to grow
• Often 100s to 1000s of different conditions
  have to be tried to succeed
• Crystals should to be a few tenth of a mm in each
  direction to be useful

21
Hanging Drop Method

• A few mL of protein solution are mixed with an
  about equal amount of reservoir solution
  containing the precipitants
• A drop of this mixture is put on a glass slide
  which covers the reservoir
• The protein/precipitant mixture in the drop is
  less concentrated than the reservoir solution so
  water evaporates from the drop into the reservoir
• The concentration of both protein and precipitant
  in the drop slowly increases leading to crystal
  formation

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Diffraction Apparatus
23
Diffraction Apparatus
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A Bigger Diffraction Apparatus
Synchrotron Light Source
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The Canadian Light Source
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Diffraction Principles (Braggs Law)
nl 2dsinq
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Diffraction Principles
Corresponding Diffraction Pattern
A string of atoms
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Protein Crystal Diffraction
Diffraction Pattern
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Diffraction Apparatus
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Converting Diffraction Data to Electron Density
F T
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Fourier Transformation
i(xyz)(hkl)
F(x,y,z) f(hkl)e d(hkl)
Converts from units of inverse space to cartesian
coordinates
32
Diffracting a Cat
Diffraction data with phase information
Real Diffraction Data
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Reconstructing a Cat
FT
Easy
FT
Hard
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The Phase Problem

• Diffraction data only records intensity, not
  phase information (half the information is
  missing)
• To reconstruct the image properly you need to
  have the phases (even approx.)
• Guess the phases (molecular replacement)
• Search phase space (direct methods)
• Bootstrap phases (isomorphous replacement)
• Uses differing wavelengths (anomolous disp.)

35
MAD X-ray Crystallography

• MAD (Multiwavelength Anomalous Dispersion
• Requires synchrotron beam lines (CLS!)
• Requires protein with multiple scattering centres
  (selenomethionine labeled)
• Allows rapid phasing
• Proteins can now be solved in just 1-2 days

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Resolution
1.2 Å
2 Å
3 Å
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Chain Tracing
Electron Chain Final Density Trace Model
38
Refinement
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Refinement
iterations
R
R S(Fo-Fc)/S(Fo)
Fc calculated structure factor
Fo observed structure factor
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The Final Result
ORIGX2 0.000000
1.000000 0.000000 0.00000
2TRX 147 ORIGX3
0.000000 0.000000 1.000000 0.00000
2TRX 148 SCALE1
0.011173 0.000000 0.004858 0.00000
2TRX 149 SCALE2
0.000000 0.019585 0.000000 0.00000
2TRX 150
SCALE3 0.000000 0.000000 0.018039
0.00000 2TRX 151
ATOM 1 N SER A 1 21.389
25.406 -4.628 1.00 23.22 2TRX 152
ATOM 2 CA SER A 1
21.628 26.691 -3.983 1.00 24.42 2TRX 153
ATOM 3 C SER A 1
20.937 26.944 -2.679 1.00 24.21 2TRX
154 ATOM 4 O SER A
1 21.072 28.079 -2.093 1.00 24.97
2TRX 155 ATOM 5 CB
SER A 1 21.117 27.770 -5.002 1.00 28.27
2TRX 156 ATOM 6
OG SER A 1 22.276 27.925 -5.861 1.00
32.61 2TRX 157 ATOM
7 N ASP A 2 20.173 26.028 -2.163
1.00 21.39 2TRX 158
ATOM 8 CA ASP A 2 19.395 26.125
-0.949 1.00 21.57 2TRX 159
ATOM 9 C ASP A 2 20.264
26.214 0.297 1.00 20.89 2TRX 160
ATOM 10 O ASP A 2
19.760 26.575 1.371 1.00 21.49 2TRX 161
ATOM 11 CB ASP A 2
18.439 24.914 -0.856 1.00 22.14 2TRX 162
A PDB coordinate file
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The PDB

• PDB - Protein Data Bank
• Established in 1971 at Brookhaven National Lab (7
  structures)
• Primary archive for macromolecular structures
  (proteins, nucleic acids, carbohydrates)
• Moved from BNL to RCSB (Research Collaboratory
  for Structural Bioinformatics) in 1998

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The PDB
http//www.rcsb.org/pdb/
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The PDB

• Contains coordinate data (primarily) from X-ray,
  NMR and modelling
• Contains files in 2 formats
• PDB format
• mmCIF (macrmolecular Crystallographic Information
  File Format)
• Contains 35,000 entries
• Currently growing exponentially

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PDB Growth
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Viewing 3D Structures

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Protein Rendering
Cylinder Ribbon (N-C gradient)
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Protein Rendering
Ribbon (2o structure)
Stick
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Protein Rendering
Space Filling Wire
Frame (Vector)
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Protein Explorer (Chime)
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Protein Explorer

• http//www.umass.edu/microbio/chime/explorer/
• Uses Chime Rasmol for its back-end
• Very flexible, user friendly, well documented,
  offers morphing, sequence structure interface,
  comparisons, context-dependent help, smart
  zooming, off-line
• Browser Plug-in (Like PDF reader)
• Compatible with Netscape (Mac Win)

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QuickPDB
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Quick PDB

• http//www.sdsc.edu/pb/Software.html
• Very simple viewing program with limited
  manipulation and very limited rendering capacity
  -- Very fast
• Java Applet (Source code available)
• Compatible with most browsers and computer
  platforms

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Rasmol
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Rasmol

• http//www.umass.edu/microbio/rasmol/
• Very simple viewing program with limited
  manipulation capacity, easy to use!
• Grand-daddy of all visual freeware
• Runs as installed stand-alone program
• Source code available
• Runs on Mac, Windows, Linux, SGI and most other
  UNIX platforms

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Conclusion

• X-ray crystallography is the primary method used
  to determine protein structures (3/4 of all
  structures in PDB)
• Has allowed determination of structures as large
  as viruses and ribosomes to be completed
• X-ray methods are fast and now depend primarily
  on computers and robots
• X-ray structures are generally more accurate than
  NMR structures, but reveal the structure in the
  solid state rather than the liquid state